Brucellosis is a global zoonotic infection and contagious illness of worldwide distribution that affects a wide spectrum of mammals, from cetaceans and pinnipeds to domestic animals and humans. The illness is caused by a gram negative bacterium that belongs to the genus Brucella which is distinguished by seven species: Brucella melitensis, B. abortus, B. suis, B. ovis, B. canis, B. neotomae and B. maris. The genus is highly homogenous in that its genomic DNA is more than 95% homologous, prompting Verger et al. to propose to reclassify the seven Brucella species as a monospecific genus (Verger J. M., Grimont F., Grimont P. A. D. and Grayon M., Brucella, a monospecific genus as shown by deoxyribonucleic acid hybridization; Int. J. Syst. Bacteriol., 1985; 35:292-5). This proposal has not yet been formally adopted. The nomenclature based on the seven species described earlier is of use because it takes into consideration the culture, host range, and epidemiological characteristics of each species.
Human brucellosis is a serious and debilitating illness, characterized by diverse clinical manifestations such as undulating fever, osteoarticular complications, endocarditic and neurological disorders. The primary pathological symptoms of the disease in cattle, goat, and sheep are abortion in pregnant females and sterility in males, due to the fact that the Brucella cells colonize the placenta, fetal tissue and reproductive organs.
Brucella is an intracellular pathogen, capable of actively invading and multiplying inside the phagocytotic cells of the host organism, mainly polymorphonuclear leukocytes (PMN) and macrophages. The Brucella exploits the phagocytic cells for transport to lymphatic tissue, uterus, and the placenta. PMN's and the macrophages fail to eliminate the bacteria from the primary site of the infection. Since the Brucella have no standard pathogenicity determinant, the exact mechanism of infection for these bacteria has not been defined.
The elimination of Brucella in the infected animal requires the generation of a cellular immune response. In vitro studies and experiments of passive transference indicate that antibodies are also involved in immune protection, especially antibodies specific to the O chain of the lipopolysaccharide (LPS).
The incidence and prevalence of brucellosis varies widely from country to country, but it can be affirmed that bovine brucellosis, caused mainly by B. abortus, is the most disperse form and is responsible for the largest economic loss (Corbel M. J., Brucellosis: an overview; Em. Inf. Dis., 1997; 3:213-21). The principal source of infection in humans is the consumption of contaminated food, especially unpasteurized milk products and contact with infected animals. Thus, prevention of human brucellosis is dependent on the control of the disease in animals.
Countries with large cattle populations employ the naturally attenuated B. abortus S-19 strain vaccine to control bovine infection. This strain is genetically stable; however, the reason for its attenuation remains unknown. The principal characteristic of this vaccine strain is its low level of pathogenicity and its elevated level of conferred protection, especially its anti-abortion effects, when the proper administration method and dosage are followed. However, the S-19 strain offers some disadvantages which include: (1) it has an elevated persistence when it is inoculated into adult animals; (2) when administered intravenously or in elevated dosages to pregnant animals it can cause abortion; (3) it is pathogenic to humans and (4) serological diagnosis is complicated because it generates agglutinating antibodies indistinguishable from those generated by pathogenic field strains. This last characteristic is due to the fact that the lipopolysaccharide of the S-19 strain, Brucella's immunodominant antigen, is identical to that of the pathogenic field strains.
Various alternatives to circumvent the disadvantages of the S-19 strain have been explored. Immunologic tests capable of discriminating between antibody titers of infected and vaccinated animals have been described. Strains that lack surface layer LPS, commonly termed as rough, have been developed. One of these strains, the mutant, rough RB51, is attenuated and incapable of producing agglutinant antibodies against LPS in the host. G. G. Schurig, et al., Biological Properties of RB51: a stable rough strain of Brucella abortus. Vet. Microbiol. 28:171-188 (1991).
The RB51 strain was derived from the pathogenic B. abortus 2308 strain by successive passages in media containing streptomycin. The genomic cause of the RB51 phenotype remains unknown. Experimental inoculations in animals demonstrated that RB51 does not interfere with serological diagnostics but the protective immunity conferred is significant but not superior to that generated by the S-19 strain. This strain was recently licensed for use in bovines.
U.S. Pat. No. 5,718,903 describes a B. abortus 2308 m106R:Tn5lacZ vaccine strain that can differentiate vaccinated from infected animals. This is a stable mutant strain that was generated by a genomic transposition which resulted in the lack of the O-antigen and which conferred protective immunity against wild B. abortus pathogenic strains.
Thus, there is a need for a Brucella vaccine that can offer superior protective immunity against brucellosis, does not interfere with the serological diagnostics of the illness, and lacks the disadvantages of the strains presently in use.